Most genes involved in human health and disease are pleiotropic: they are expressed in diverse cell types, act at various developmental stages, or participate in different developmental pathways. These pleiotropic effects can slow the analysis of human disease gene function; for example, if a gene has both an early and a late function, a knock-out mutation can block development at the early stage which prevents discovery or analysis of the later function. Investigation of mutations in zebrafish genes provides a way to solve this problem. Due to an ancient genome duplication event, zebrafish possess two copies of many single-copy human disease genes. During the course of evolution, gene functions present in the last common ancestor of zebrafish and human have frequently assorted to different copies of the zebrafish duplicates of human genes (subfunctionalization). For example, the early function might go to one copy and the late function to the other. Analysis of mutations in both genes can more fully or more easily reveal gene functions conserved among all vertebrates. The general goal of the proposed work is to contribute to our understanding of the functions of human disease genes by identifying zebrafish duplicate co-orthologs of human disease genes and making mutations in high priority genes that will be freely available to the research community. Specific Aim 1 is to identify all duplicated genes from the zebrafish sequencing project and note which ones are co-orthologs of human disease genes. Specific Aim 2 is to conduct a genotype-driven screen to produce mutations in both zebrafish copies of human disease genes deemed of high priority by the research community. The mutations will be posted immediately on a website and will be freely available on request. Specific Aim 3 is to analyze mutations in genes involved in diseases of the neural crest and in neural patterning. This project is important because the mutations will provide a lasting resource for the analysis of disease genes. Furthermore, the coordinated and simultaneous functional investigation of both copies of human disease genes will reveal conserved gene functions that may be obscured by analysis of the single gene in tetrapods.